Problems related to low oral bioavailability due to poor solubility of new drug candidates are an increasing challenge in pharmaceutical research and formulation development. One efficient way to improve solubility is the utilization of nanocrystallization techniques: pharmaceutical nanocrystals are solid drug particles covered by a stabilizer layer with approximated size typically between 100 and 500 nm. Nanocrystal studies have been conducted since the beginning of the 1990’s and the first product entered the market after 10 years of intensive research. At first, nanocrystals were utilized purely for improved dissolution, but today also controlled release applications are in use.

Applications

The most important benefit with nanocrystals is the increased surface area, which speeds up the dissolution [1]. Another, but often forgotten impact is the higher saturated solubility with nanosized particles [2]. Accordingly, nanocrystals are mostly utilized for improved dissolution of poorly soluble drug materials [3]. However, during the last years utilization of nanocrystals in controlled drug release applications has been studied more and more [4]. Controlled release with nanocrystals can be reached for example by tailoring the particle size of very poorly soluble drug material or by attaching nanocrystals inside a matrix structure.

Production

Nanocrystals can be produced by top-down methods, e.g. milling or high-pressure homogenization of larger particles or by bottom-up techniques, e.g. building up larger particles molecule by molecule. In our research group we are mainly producing nanocrystals by wet ball milling [5], though we have also utilized anti-solvent precipitation in some studies [4]. In our laboratory, we have the capability to produce small scale batches containing drug material from 100 mg to 8 g.

Challenges

Often nanocrystals are easy to produce, and instead the stability and state after further processing are the main challenges [6]. The smaller the nanocrystals are, the higher their tendency for aggregation. Another challenge is to successfully turn the in vitro enhancement in drug release to improved bioavailability in vivo [7]. The selection of stabilizer is also a crucial step, as well as the overall composition of the formulation.

Future work

The research with nanocrystals in Helsinki is mainly focused in two areas: 1) interactions between the drug and stabilizer material in order to rationalize the selection method for a successful stabilizer for a certain drug, and 2) the role of stabilizer in improving the bioavailability of nanocrystalline formulations.

PSSRC Facilities

The nanocrystallization research group in Helsinki headed by docent Leena Peltonen has strong know-how in physicochemical characterization of nanocrystals and systematic understanding of process parameters on the milling outcome. We are mainly studying the milling process for nanocrystal production. Recently, we have started studies with impact of different stabilizers on cell uptake and permeability properties and we are utilizing some new techniques, like CARS, in co-operation with Professor Clare Strachan in this research area.

Non-linear Optical Imaging

Non-linear optical imaging is an emerging technique for imaging drugs and dosage forms [1]. Non-linear optical imaging may be used for non-destructive, non-contact imaging of solid drugs and dosage forms. It offers chemical and structural specificity with no requirement for labels, sub-micron spatial resolution (inherent confocal nature), rapid video-rate image acquisition, and the ability to image samples in aqueous environments in situ.

These combined features make non-linear optical imaging unique compared to existing imaging approaches in the pharmaceutical setting and make the technique well suited to a wide range of solid-state formulation and drug delivery analyses. These include imaging chemical and solid-state form distributions in dosage forms, drug release and dosage form digestion, and drug and micro/nanoparticle distribution in tissues and within live cells. While non-linear optical imaging is comparatively well established in the biomedical field, pharmaceutical applications of non-linear optical imaging are much less widely explored.

Principle of Non-linear Optical Imaging

Non-linear optical imaging involves irradiation of a sample with laser light (at one or two wavelengths) through an optical microscope and detection of scattered light at a different frequency. Non-linear optical imaging is also sometimes referred to as multi-photon imaging since the non-linear processes involve several photons (Figure 1). The technique encompasses a range of non-linear optical phenomena including second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (TPF). In SHG, the energy of two photons is combined to emit light at half the laser wavelength. This process depends on the structural symmetry of the sample, and can be used to resolve crystalline and amorphous materials and some different polymorphic forms. In CARS, three photons at two or three wavelengths interact to efficiently generate light at a shorter wavelength (anti-Stokes Raman scattering). The technique is related to normal (spontaneous) Raman imaging, and is also used for label-free chemically-selective imaging. However CARS imaging is orders of magnitude faster, the spatial resolution is usually better and interference from fluorescence may be avoided. TPF is related to normal (one-photon) fluorescence, but it involves the energy of two incident photons instead of one with the advantage of being inherently confocal. Some materials (e.g. indomethacin, doxorubicin) generate TPF and so can be imaged with this technique without the requirement for labels. Vibrational energy level diagrams representing the SHG, CARS and TPF processes are shown in Figure 1.

Since the non-linear optical phenomena have different advantages and specificities, it is often very helpful to collect a combination of these signals at the same time with the same imaging setup. This is known as "multi-modal" imaging.

Imaging Solid Drugs and Dosage Forms

It is becoming widely recognised that critical solid dosage form properties, such as drug dissolution and release, are dependent not only on the formulation composition but also the component and solid state form distribution. Non-linear optical imaging is well suited to imaging a range of dosage forms. It is capable of rapidly imaging different chemical components and solid forms with high resolution (micron or sub-micron) in three dimensions. In general the data for the images may be collected in a few seconds or less. The technique may also be used to image changes in dosage forms in situ during drug release/dissolution and storage [2].

Distributions of components in tablets may be imaged in 2D or 3D, as shown in Figures 2 and 3. Both drug and excipient distributions may be imaged.

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Future Work

As mentioned above, the technique is suited for real time imaging of drug release/dissolution. In collaboration with the Optical Sciences Group, University of Twente, The Netherlands and Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University, Duesseldorf, Germany we are currently working on imaging drug and dosage form changes in a flow-through cell while simultaneously analysing drug concentration in solution.

Non-linear optical imaging is also well suited to real-time imaging of cells and tissues. We are currently working on imaging delivery of poorly water soluble drugs in various types of formulations in vitro and in vivo. If feasible, this approach will facilitate bringing together the analysis of drug release/dissolution and permeability, and should help lead to better understanding of absorption of these drugs.

PSSRC Facilities

Asst. Prof. Clare Strachan (Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki) has several years' experience in non-linear optical imaging of a range of solid dosage forms. A fully integrated commercial non-linear optical microscope (Leica TCS SP8 CARS microscope) is available at the University of Helsinki. This is the first commercially available fully integrated CARS microscope in the world. The microscope uses a picosecond solid-state-laser light source to excite single Raman lines within a range of 1250 cm-1 to 3200 cm-1 for CARS imaging. It gives access to molecular specific contrast based on a variety of Raman-active vibrations relevant to pharmaceutical applications. Second harmonic generation (SHG) and two-photon fluorescence (TPF) are also possible with the setup. The microscope is also capable of one-photon fluorescent imaging in the UV and visible wavelengths. All non-linear and fluorescence phenomena can be imaged on exactly the same sample with the same microscope, and therefore a direct comparison of the imaging approaches can be made.